Home › Biochemistry › iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
Biochemistry JoVE (Open Access) Citable · DOI

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

DOI: 10.3791/2638-v
What you'll learn
  • ✓ Perform UV crosslinking of RNA-binding proteins in cultured cells
  • ✓ Execute immunoprecipitation and RNA labeling workflow for iCLIP
  • ✓ Analyze genome-wide protein-RNA binding sites at nucleotide resolution
  • ✓ Purify and prepare cDNA libraries for sequencing
Protocol

Biopharma Insights The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

Difficulty
advanced
Total time
~3–4 days (includes UV crosslinking, IP, labeling, gel purification, and library prep)
Model organism
Tissue culture cells (protocol-agnostic; HEK293 or similar)
Biosafety
BSL-1

Steps

1
UV crosslink RNA-binding proteins in tissue culture cells

Expose cultured cells to UV light to covalently crosslink RNA-binding proteins to their target RNA molecules. This step preserves protein-RNA interaction sites for downstream mapping.

▶ 01:19
2
Prepare magnetic beads for immunoprecipitation

Wash and prepare magnetic beads coated with antibodies or binding reagents to enable efficient capture of the target RNA-binding protein complex.

▶ 02:10
3
Lyse cells and perform partial RNA digestion

Lyse crosslinked cells and use RNase to digest RNA non-specifically, leaving only the protected RNA fragment directly bound to the protein of interest.

▶ 02:53
4
Immunoprecipitate RNA-binding protein complexes

Use prepared magnetic beads to capture the target RNA-binding protein and its bound RNA fragments from the cell lysate via antibody-mediated pulldown.

▶ 03:47
5
Label RNA 5' end with radioactive or fluorescent marker

Phosphorylate and label the 5' end of protected RNA fragments to enable sensitive detection and visualization during gel-based purification steps.

▶ 04:26
6
Separate protein-RNA complex by SDS-PAGE and transfer

Denature and separate the crosslinked protein-RNA complex on a polyacrylamide gel, then transfer to a membrane for visualization and extraction.

▶ 05:08
7
Purify cDNA by gel excision and reverse transcription

Excise and isolate the labeled RNA band from the membrane, reverse transcribe to cDNA, and purify for library construction and next-generation sequencing.

▶ 06:27
8
Interpret representative results and validate mapping

Review example iCLIP data showing genome-wide distribution of protein-RNA binding sites, confirming nucleotide-level resolution and biological reproducibility.

▶ 08:44
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